Interpretive Summary: This paper describes a functional genomics study of an ATP/ADP translocase in Candidatus Liberibacter asiaticus (Las), the phloem-restricted, gram-negative alphabacterium that causes the devastating disease of citrus known as huanglongbing. A single translocase encoding gene was identified through an in silico analysis of the recently completed Las genome. Bacterial ATP/ADP translocases transport ATP from the host cell cytosol, across the lipid bilayer, and into the bacterial cell. This ability to import ATP directly allows bacteria that posses this enzyme to act as “energy parasites” and uptake ATP produced by their hosts.
The protein product of the translocase gene demonstrated characteristics typical for members in this family of proteins, including 12 transmembrane helices and an isoelectric point of 9.4. Due to an inability to genetically manipulate Las at the present time, the translocase (tlc) open reading frame was cloned and expressed in Escherichia coli to determine if this putative gene was in fact functional. Uptake studies using radiolabeled ATP demonstrated that the translocase could indeed transport exogenous ATP into the cell. Subsequent competition assays revealed that the system was highly specific for ATP and ADP but not other ribonucleotides. In addition, the importance of the translocase was exemplified by its presence in the related Liberibacter species known as Ca. Liberibacter solanacearum (Lso). Although the bacterial translocase has been characterized in other intercellular pathogens such as Chlamydia trachomatis and Rickettsia prowazeki, this is the first characterization of an ATP/ADP translocases in an obligate-intracellular plant pathogen and, given the conservation of the ATP uptake mechanism, may represent an evolutionary link between plant and animal pathogens. In addition, because of its single loci and important function, the translocase may represent a novel target in which to combat this deadly disease.

Technical Abstract:
ATP/ADP translocases allow for the transport of ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate-intracellular bacteria. Of the bacterial ATP/ADP translocases characterized thus far, all have been found in endosymbionts of protozoa or pathogens of higher order animals including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen, Candidatus Liberibacter asiaticus (Las), the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The Las gene encoding the translocase (tlc) was subsequently expressed in Escherichia coli and shown to enable E.coli to uptake ATP directly into the cell. Competition assays in the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but other nucleotides, if present in high concentrations, could also be uptaken and/or block the ability of the translocase to uptake ATP. Using the amino acid sequence from the Las encoded translocase, a homologous protein was identified in Ca. Liberibacter solanacearum (Lso), the bacterium associated with zebra chip disease. This is the first reported characterization of an ATP/ADP translocase from a plant pathogen. The presence of this conserved mechanism for ATP uptake in both plant and animal pathogens may serve as an evolutionary link between the two.